In laser scanning microscopy, an area of an object is illuminated in a raster-like manner and scanned by a laser beam point by point. In most cases, a parallel laser beam, which is typically 10 mm in diameter, is deflected for illumination according to a desired pattern, about a deflection angle—e.g. in a similar manner as an electron beam in a Braun tube—by using a deflecting device. The deflected laser beam is then focussed by an optical system in an intermediate image plane of the laser scanning microscope and subsequently imaged by an objective onto or into the object.
The focused laser beam interacts with the object, with reflected radiation or fluorescence radiation being generated, which returns along the same path taken by the laser beam during illumination. Said radiation is then deflected into a detector beam path by a beam splitter arranged, in most cases, preceding the deflecting device in the illumination direction, there being arranged, within the detector beam path, at least one imaging system which focusses the detected beam in a further image plane. Depending on the particular application, there may be several detector beam paths, each receiving a predetermined spectral range of the radiation coming from the object.
The deflecting device causes a raster-like movement of the focussed laser beam over the object surface area, with information on the present state of the deflection, i.e. on the present position of the focussed laser beam in the object surface area, being required in order to correctly assign, at this point in time, the radiation received in the detector beam path to an image point. Thus, the precision of said deflection affects the geometric correspondence of the scanned image and the object; in this connection, one also speaks of image linearity.
In this case, the problem arises that, the deflecting device, which may be realized, for example, in the form of two tiltable mirrors, follows a predetermined deflection behaviour, which is usually determined by a control signal, only in a more or less precise manner due to inertia and various disturbances. Since, at the same time, a high operating speed, i.e. a high scanning frequency, is desired for the deflecting device, the deflecting device needs to satisfy certain minimum requirements in order to guide the focussed laser beam over the object surface area at a constant high speed in a precise manner.
Naturally, in doing so, a characteristic as linear as possible is pursued for the deflecting device. High scanning frequencies may be achieved in a particularly easy manner, if a reciprocating movement of the laser beam over the object is utilized for scanning. This is referred to as bidirectional scanning. For this purpose, for example, each tiltable mirror of a deflecting device comprising two tiltable mirrors is usually controlled via a triangular signal. Thus, one tiltable mirror effects deflection along a line of the object surface area, with one such line corresponding to a half-cycle of the triangular signal, e.g. from the minimum value to the next maximum value, and the other tiltable mirror deflects perpendicular to the line direction, with the aforementioned half cycle of the triangular signal for this tiltable mirror corresponding to a passage of the laser beam over the entire object surface area, for example, from top to bottom. Of course, the second tiltable mirror, which is required to move much more slowly, may also be controlled by a sawtooth signal.
The phases and amplitudes of the control signal, which is thus composed of two triangular signals, accordingly have a direct effect on the linearity of the movement of the focussed laser beam over the object surface area. However, in this connection, it is mandatory that the deflecting device, e.g. the aforementioned tiltable mirrors, exactly follow the control signal, which, however, is hardly ever the case. For example, if oscillating mirrors are used in the deflecting device, as is common practice in laser scanning microscopes, said mirrors can follow a triangular signal only to a limited extent. Moreover, triangular signals may be regarded as the Fourier synthesis of odd harmonics (multiples) of the deflection frequency. However, unavoidable phase delays and lower transfer factors of higher harmonics result in non-linear movements of the tiltable mirrors.
In order to increase the precision with which the actual deflection by the deflecting device follows a desired movement, DE 197 02 752 A1 suggests to provide a feedback device on the deflecting device in order to measure the position of the tiltable mirrors used in the deflecting device. The condition of the deflecting device is thus detected and used in combination with an anticipatory control for open-loop control of desired characteristics.
In order to determine the dynamic characteristics of the deflecting device, the deflecting device is first controlled with pure sinus signals, with a wide frequency range being swept and the amplitude and phase of the movement of the tiltable mirrors being measured. Using a Fourier series, a control signal may then be synthesized by including, in the individual Fourier coefficients, the phase rotation of the frequency of the respective coefficient as an offset and the inverse value of a transmission factor of the respective coefficient of the deflecting device for the corresponding frequency as part of the amplitude. In this way, the control signal is predistorted such that the movement of the deflecting device ultimately approaches the desired movement as closely as possible.
However, since the deflecting device comprising the control device for controlling cannot be considered to be a linear system in this case, so that linear superposition of the aforementioned Fourier coefficients accordingly does not lead to an optimal result for said movement, according to DE 197 02 752 A1, any possible residual error remaining is determined for the individual coefficients and compensated for by a further predistortion of the control signal, in a third step reverting to the feedback device.
This method, which involves extensive calculations, achieves high precision at deflection frequencies below 1 kHz. In order to obtain full correspondence of the forwardly scanned and backwardly scanned lines in the bidirectional scan, the movements, however, have to be symmetrical with respect to the points of reversal. There is, thus, the problem that, due to a lack of symmetry, an offset between the image lines picked up during a forward rotation of the tiltable mirrors and the image lines picked up during a countercurrent backward rotation cannot be avoided. Such offset limits the admissible deflection frequency, so that, for the time required to take a picture, which is naturally desired to be kept as short as possible, a lower limit is set, if image linearity is not to be adversely affected. For an image format of 512×512 pixels, it has turned out that an offset of 0.2 pixels for adjacent image lines can just be tolerated.